The goal of the current project is to determine the cell-mediated signals that direct cell patterning of the developing inner ear using an in vitro assay. Preliminary data from this lab revealed that dissociated, embryonic inner ear cells reassociate into three distinct aggregate types in vitro, yet only one aggregate type contains sensory hair cells. These hair cells are surrounded by supporting cells, similar to the pattern observed in vivo. How this precise reassoication occurs is unknown. Aim 1 will further characterize the aggregation of inner ear cells in vitro by testing the hypotheses that both cochlear and vestibular hair cells reaggregate and that this reaggregation is limited to early stages of development. Aims 1 and 2 will test two alternative mechanisms by which inner ear cells organize into the precise mosaic. Aim 2 will test the hypothesis that individual hair cells differentiate, then migrate within the cultures to aggregate into specific patterns using cell recognition cues. Aim 3 will test the hypothesis that individual hair cells remain undifferentiated until aggregates are formed, then use the notch signaling pathways to establish cell fate. Together, these experiments will provide information regarding the mechanisms used for organizing normal auditory and vestibular epithelia and may provide insight into means of treating congenital anomalies of the inner ear.